RESUMO
BACKGROUND: Hyphomicrobium nitrativorans strain NL23 and Methylophaga nitratireducenticrescens strain JAM1 are the principal bacteria involved in the denitrifying activities of a methanol-fed, fluidized-bed marine denitrification system. Strain NL23 possesses the complete denitrification pathway, but cannot grow under marine conditions in pure cultures. Strain JAM1 is a marine bacterium that lacks genes encoding a dissimilatory nitrite (NO2 -) reductase and therefore cannot reduce NO2 -. Here, we report the characterization of some of their physiological traits that could influence their co-habitation. We also perform co-cultures to assess the potential synergy between the two strains under marine and denitrifying conditions. METHODOLOGY: Anoxic planktonic pure cultures of both strains were grown with different concentrations of nitrate (NO3 -). Anoxic planktonic co-cultures could only be cultured on low NaCl concentrations for strain NL23 to grow. Biofilm co-cultures were achieved in a 500-mL bioreactor, and operated under denitrifying conditions with increasing concentrations of NaCl. NO3 - and NO2 - concentrations and the protein content were measured to derive the denitrification rates. The concentrations of both strains in co-cultures were determined by quantitative PCR (qPCR). Ectoine concentration was measured by mass spectrometry in the biofilm co-culture. The biofilm was visualized by fluorescence in situ hybridization. Reverse-transcription-qPCR and RNA-seq approaches were used to assess changes in the expression profiles of genes involved in the nitrogen pathways in the biofilm cultures. RESULTS: Planktonic pure cultures of strain JAM1 had a readiness to reduce NO3 - with no lag phase for growth in contrast to pure cultures of strain NL23, which had a 2-3 days lag phase before NO3 - starts to be consumed and growth to occur. Compared to strain NL23, strain JAM1 has a higher µmax for growth and higher specific NO3 - reduction rates. Denitrification rates were twice higher in the planktonic co-cultures than those measured in strain NL23 pure cultures. The biofilm co-cultures showed sustained denitrifying activities and surface colonization by both strains under marine conditions. Increase in ectoine concentrations was observed in the biofilm co-culture with the increase of NaCl concentrations. Changes in the relative transcript levels were observed in the biofilm culture with genes encoding NapA and NapGH in strain NL23. The type of medium had a great impact on the expression of genes involved in the N-assimilation pathways in both strains. CONCLUSIONS: These results illustrate the capacity of both strains to act together in performing sustainable denitrifying activities under marine conditions. Although strain JAM1 did not contribute in better specific denitrifying activities in the biofilm co-cultures, its presence helped strain NL23 to acclimate to medium with NaCl concentrations >1.0%.
RESUMO
BACKGROUND: Methylophaga nitratireducenticrescens strain JAM1 is a methylotrophic, marine bacterium that was isolated from a denitrification reactor treating a closed-circuit seawater aquarium. It can sustain growth under anoxic conditions by reducing nitrate ([Formula: see text]) to nitrite ([Formula: see text]). These physiological traits are attributed to gene clusters that encode two dissimilatory nitrate reductases (Nar). Strain JAM1 also contains gene clusters encoding two nitric oxide (NO) reductases and one nitrous oxide (N2O) reductase, suggesting that NO and N2O can be reduced by strain JAM1. Here we characterized further the denitrifying activities of M. nitratireducenticrescens JAM1. METHODS: Series of oxic and anoxic cultures of strain JAM1 were performed with N2O, [Formula: see text] or sodium nitroprusside, and growth and N2O, [Formula: see text], [Formula: see text] and N2 concentrations were measured. Ammonium ([Formula: see text])-free cultures were also tested to assess the dynamics of N2O, [Formula: see text] and [Formula: see text]. Isotopic labeling of N2O was performed in 15NH4+-amended cultures. Cultures with the JAM1ΔnarG1narG2 double mutant were performed to assess the involvement of the Nar systems on N2O production. Finally, RT-qPCR was used to measure the gene expression levels of the denitrification genes cytochrome bc-type nitric oxide reductase (cnorB1 and cnorB2) and nitrous oxide reductase (nosZ), and also nnrS and norR that encode NO-sensitive regulators. RESULTS: Strain JAM1 can reduce NO to N2O and N2O to N2 and can sustain growth under anoxic conditions by reducing N2O as the sole electron acceptor. Although strain JAM1 lacks a gene encoding a dissimilatory [Formula: see text] reductase, [Formula: see text]-amended cultures produce N2O, representing up to 6% of the N-input. [Formula: see text] was shown to be the key intermediate of this production process. Upregulation in the expression of cnorB1, cnorB2, nnrS and norR during the growth and the N2O accumulation phases suggests NO production in strain JAM1 cultures. DISCUSSION: By showing that all the three denitrification reductases are active, this demonstrates that M. nitratireducenticrescens JAM1 is one of many bacteria species that maintain genes associated primarily with denitrification, but not necessarily related to the maintenance of the entire pathway. The reason to maintain such an incomplete pathway could be related to the specific role of strain JAM1 in the denitrifying biofilm of the denitrification reactor from which it originates. The production of N2O in strain JAM1 did not involve Nar, contrary to what was demonstrated in Escherichia coli. M. nitratireducenticrescens JAM1 is the only reported Methylophaga species that has the capacity to grow under anoxic conditions by using [Formula: see text] and N2O as sole electron acceptors for its growth. It is also one of a few marine methylotrophs that is studied at the physiological and genetic levels in relation to its capacity to perform denitrifying activities.
